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α

β

α α α

α α

β β

αα

α β

αβ

β α

TM

CY HLA class I

α1α2

α3 β2Μ

pep#de4 3 333

5$amino$acids 3

Numbers+of+HLA+Alleles+HLA$Class$I$Alleles$ 7,089$HLA$Class$II$Alleles$ 2,065$HLA$Alleles$ 9,154$HLA+Class+I+Gene+ A+ B+ C+Alleles$ 2,244$ 2,934$ 1,788$Proteins$ 1,612$ 2,211$ 1,280$Nulls$ 109$ 97$ 47$HLA+Class+II+Gene+ DRB+ DQB1+ DPB1+Alleles$ 1,418$ 323$ 185$Proteins$ 1,051$ 216$ 153$Nulls$ 32$ 7$ 6$

IMGT/HLA$database$Version$Report$I$3.12$(2013I04)

• • • • • • • 

sequence+specific+oligonucleo#de+5’UTR ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 3’UTR

B*51:02:01((B*58:01:01(

5’UTR ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 3’UTRsequencing$based$typing +

CombinaSon$1$$$$B*07:021(+(B*35:011(CombinaSon$2$$$$B*07:18(+(B*35:05CombinaSon$3$$$$B*07:09(+(B*35:34CombinaSon$4$$$$B*07:24(+(B*35:15

Ambiguity

HLA-A HLA-C HLA-B HLA-DRB1 HLA-DQB1 HLA-DPB1

Locus+ length+(bp)+HLA2A( 3,398$HLA2C( 4,296$HLA2B( 4,440$

HLA2DRB1( 11,899$HLA2DQB1( 7,118$HLA2DPB1( 13,605$

$$$$$$$$$$$$$$$$HLA2 ((((($$$M((((((((((((A((((((((((((C((((((((((((B(((((((((DRB1((((DQB1(((((DPB1

locus Length$(bp)

HLAIA$ 3,398$

HLAIB$ 4,296$

HLAIC$ 4,440$

HLAIDRB1$ 11,899$

HLAIDQB1$ 7,118$

HLAIDPB1$ 13,605$

$

Nextera$DNA$Sample$PreparaSon$Kit$(illumina)

$

Read$length $2$x$250$bp$Output $ $7.5I8.5$Gb$Total$Sme $39$hr$

HLA-B

Sequence reads

Alignment

Diplotype sequence

SNVs

ATG!TAC

CAA!TTGCAA!TTG

TAC!AGGTAC!AGG

T!C

maximum$differences$=$80$in$the$read(250bp)

Heterozygous$SNVs

HLA-B

Sequence reads

Alignment

Diplotype sequence

SNVs

ATG!TAC

CAA!TTGCAA!TTG

TAC!AGGTAC!AGG

T!C

HLA-B

Sequence reads

Alignment

Diplotype sequence

SNVs

250bp600bp

800bp1000bp

ATG!!!!TAC

CAA!!!!TTG

CAA!!!!TTG

TAC!!!!AGG

TAC!!!!AGG

T!!!!C

Agarose+gel+size+selec#on

HLA-B

Sequence reads

Alignment

Diplotype sequence

SNVs

250bp600bp

800bp1000bp

ATG!!!!TAC

CAA!!!!TTG

CAA!!!!TTG

TAC!!!!AGG

TAC!!!!AGG

T!!!!C

HLA-B

Sequence reads

Alignment

Haplotype sequences

SNVs

250bp600bp

800bp1000bp

!ATG!!!!TAC

CAA!!!!TTG

TAC!!!!AGG

T!!!!C

HLA$haplotype$sequence$2HLA$haplotype$sequence$1

B*07:02:01

HLA-B ex7 ex6 ex5 ex4 ex3 ex2 ex1

Phased haplotype 1

Phased haplotype 2

Before phasing

B*51:01:01

B*15:02:01

Haplotype$sequence

Alignment$to$HLA$gene$sequence$of$hg19$reference$BWA,+Samtools,+Picard+

Detect$SNVs$and$Indels$UnifiedGenotyper+in+GATK+

Phase$haplotype$using$heterozygous$SNVs$on$PE$reads$Perl+script+

Divide$alignment$into$two$phased$one$as$haplotype$Perl+script+

PairedIread$fastq$hg19$reference$ maximum$differences$=$80

Create$consensus$sequence$FastaAlternateReferenceMaker+in+GATK,+Perl+script+

Sample+ID+ PCRQSSO+ NextQgen+SBT+ Average+read+depth+(allele1+/+allele2)+Determined+sequence+(%)+

(allele1+/+allele2)+1$ B*51:01:01( B*15:02( B*51:01:01( B*15:02:01( 2593.5$/$2431.3$ 100+/+100+2$ B*07:02:01( B*15:02( B*07:02:01( B*15:02:01( 3391.2$/$4206.2$ 100+/+100+3$ B*35:01:01( B*15:02( B*35:01:01:01( B*15:02:01( 2096.2$/$2431.8$ 100+/+100+4$ B*15:21( B*15:02:01( B*15:21( B*15:02:01( 4308.2$/$4693.6$ 100+/+100+5$ B*35:05( B*15:02:01( B*35:05:01( B*15:02:01( 4264.3$/$3620.2$ 100+/+100+6$ B*15:18( B*15:02( B*15:18:01( B*15:02:01( 1239.9$/$1277.3$ 100+/+100+7$ B*40:06:01:01( B*15:02( B*40:06:01:01( B*15:02:01( 5552.1$/$5139.5$ 100+/+100+8$ B*52:01:01( B*15:02( B*52:01:01:01( B*15:02:01( 4651.7$/$5085.9$ 100+/+100+9$ B*15:01:01:01( B*57:01:01( B*15:01:01:01( B*57:01:01( 2326.4$/$2195.1$ 100+/+100+10$ B*54:01( B*57:01:01( B*54:01:01( B*57:01:01( 1999.6$/$2034.6$ 100+/+100+11$ B*40:06:01:01( B*57:01:01( B*40:06:01:01( B*57:01:01( 3087.4$/$2833.9$ 100+/+100+12$ B*15:11:01( B*57:01:01( B*15:11:01( B*57:01:01( 1948.4$/$1721.9$ 100+/+100+13$ B*15:01:01:01( B*57:01:01( B*15:01:01:01( B*57:01:01( 1660.0$/$1482.0$ 100+/+100+14$ B*44:03:01( B*57:01:01( B*44:03:01( B*57:01:01( 806.0$/$668.2$ 100+/+100+15$ B*55:07( B*58:01( B*55:07( B*58:01:01( 771.6$/$1000.2$ 100+/+100+16$ B*38:01:01( B*58:01( B*38:01:01( B*58:01:01( 1565.1$/$2139.7$ 100+/+100+17$ B*48:01:01( B*58:01( B*48:01:01( B*58:01:01( 5555.3$/$4610.8$ 100+/+100+18$ B*35:01:01( B*58:01:01( B*35:01:01:01( B*58:01:01( 819.5$/$778.1$ 100+/+100+19$ B*15:25:01( B*58:01:01( B*15:25:01( B*58:01:01( 3441.2$/$3852.0$ 100+/+100+20$ B*54:01( B*58:01:01( B*54:01:01( B*58:01:01( 1308.3$/$1454.1$ 100+/+100+21$ B*51:02:01( B*58:01:01( B*51:02:01( B*58:01:01( 1464.0$/$1132.7$ 100+/+100+22$ B*51:01:01( B*58:01:01( B*51:01:01( B*58:01:01( 462.9$/$636.3$ 100+/+100+23$ B*51:02:01( B*58:01:01( B*51:02:01( B*58:01:01( 637.0$/$663.7$ 100+/+100+24$ B*39:23( B*58:01:01( B*39:23( B*58:01:01( 2112.6$/$2553.3$ 100+/+100+

$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$HLAIB$ex7$ex6$$$$$$$$$$$ex5$$$ex4$$$$$$$$$$$$$$$$$$$$$$$$ex3$$$$$$$$$$$ex2$$$$$ex1

Haplotype$sequence

Alignment$to$HLA$gene$sequence$of$hg19$reference$BWA,+Samtools,+Picard+

Detect$SNVs$and$Indels$UnifiedGenotyper+in+GATK+

Phase$haplotype$using$heterozygous$SNVs$on$PE$reads$Perl+script+

Divide$alignment$into$two$phased$one$as$haplotype$Perl+script+

PairedIread$fastq$hg19$reference$ maximum$differences$=$80

Create$consensus$sequence$FastaAlternateReferenceMaker+in+GATK,+Perl+script+

•  pIgalaxy$hbp://pIgalaxy.ddbj.nig.ac.jp$

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ex1ex2ex3ex4ex5ex6ex7

Low$depth Low$depth

GC$contents$(%)

CCCCTCTGCCCCACCCACCCCCAGACCCGCCACCCCACCCAC$78.6%$GC

(A) (B)

72oC 3min98oC 30sec98oC 10sec63oC 30sec 7cycles72oC 3min10oC

55oC 5min10oC

PCR (10ng) 4ulTD 5ulTDE1 1ul

Index1 primer 1ulIndex2 primer 1ul2×Kapa HiFi HS RM 5ulPPC 1ulTagmentation DNA 2ul

(A) (B)

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5’ CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG

5’ AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC

51bp 47bp

0.2x$diluted$beads$0.1x$diluted$beads$

0.05x$diluted$beads$

R2=0.99749

AMPure$XP$volume$(ul)

Bound$DNA$(pg)$

•  20%$PEG8000$and$2.5M$NaCl$– 10g$polyethylene$glycol$(PEG)$8000$– 7.3g$NaCl$–  the$volume$to$50$ml$with$MilliQ$water$

$$$$$$AMPure$beads$1,000ml$$

$ $$$$$$$Centrifuge$5,000rpm$5min$$

$ $ $$$$$$$Supernatant$1,000ml$$$$$$$$Beads $$

$ $ $$$$$$20%$PEG$500ml$(Supernatant$volume)$$$$$$$AMPure$20%$PEG$beads$

AMPure$beads$volume$raSo$

Beads$raSo$ Selected$length$1.2x$ >100bp$0.8x$$ >200bp$0.6x$ >300bp$0.5x$ >500bp$0.45x$ >600bp$0.35x$ >1500bp$

Target$fracSon

>1000bp$selecSon$>500bp$selecSon$

500I1000bp$selecSon

Target$fracSon

>1000bp$selecSon$

>500bp$selecSon$

500I1000bp$selecSon

0.2x$diluted$beads$

0.1x$diluted$beads$

0.05x$diluted$beads$

Collaborators$and$Acknowledgments$